Activation of plant immunity by exposure to dinitrogen pentoxide gas generated from air using plasma technology

Reactive nitrogen species (RNS) play an important role in plant immunity as signaling factors. We previously developed a plasma technology to partially convert air molecules into dinitrogen pentoxide (N2O5), an RNS whose physiological action is poorly understood. To reveal the function of N2O5 gas in plant immunity, Arabidopsis thaliana was exposed to plasma-generated N2O5 gas once (20 s) per day for 3 days, and inoculated with Botrytis cinerea, Pseudomonas syringae pv. tomato DC3000 (Pst), or cucumber mosaic virus strain yellow (CMV(Y)) at 24 h after the final N2O5 gas exposure. Lesion size with B. cinerea infection was significantly (P < 0.05) reduced by exposure to N2O5 gas. Propagation of CMV(Y) was suppressed in plants exposed to N2O5 gas compared with plants exposed to the air control. However, proliferation of Pst in the N2O5-gas-exposed plants was almost the same as in the air control plants. These results suggested that N2O5 gas exposure could control plant disease depending on the type of pathogen. Furthermore, changes in gene expression at 24 h after the final N2O5 gas exposure were analyzed by RNA-Seq. Based on the gene ontology analysis, jasmonic acid and ethylene signaling pathways were activated by exposure of Arabidopsis plants to N2O5 gas. A time course experiment with qRT-PCR revealed that the mRNA expression of the transcription factor genes, WRKY25, WRKY26, WRKY33, and genes for tryptophan metabolic enzymes, CYP71A12, CYP71A13, PEN2, and PAD3, was transiently induced by exposure to N2O5 gas once for 20 s peaking at 1–3 h post-exposure. However, the expression of PDF1.2 was enhanced beginning from 6 h after exposure and its high expression was maintained until 24–48 h later. Thus, enhanced tryptophan metabolism leading to the synthesis of antimicrobial substances such as camalexin and antimicrobial peptides might have contributed to the N2O5-gas-induced disease resistance.


Introduction
Developing agricultural systems that minimize environmental impacts remains a major challenge. Excessive use of chemical fertilizers and pesticides include the risks of contaminating the soil and harming the ecosystem [1]. The concept of Integrated Pest Management (IPM), which effectively combines a variety of control strategies rather than relying solely on chemical pesticides, has become widely accepted in the context of pest control [1]. These efforts can contribute to the achievement of the United Nation's Sustainable Development Goals (SDGs). Therefore, it is desirable to develop further technologies to reduce the environmental impacts of agriculture and realize sustainable agricultural systems. Plasma is a state of matter that can be observed as lightning or auroras in nature and is characterized by electrically charged energetic particles that can form highly reactive states such as radicals. Atmospheric pressure air plasma can be generated using air under atmospheric pressure with low electric power (<100 W) potentially supplied by renewable energy resources. Because of the low resource demands for its generation as well as its ability to generate biologically active reactive oxygen species (ROS) and reactive nitrogen species (RNS) [2], atmospheric pressure air plasma is attracting attention as a potentially sustainable technology in the fields of medicine and agriculture [3,4].
Typical reactive species produced in atmospheric pressure air plasma include ozone (O 3 ) as a ROS as well as nitric oxide (NO), nitrogen dioxide (NO 2 ), and dinitrogen pentoxide (N 2 O 5 ) as RNS [5][6][7]. It is well known that ROS and RNS are important signaling factors in the immune responses of plants. Plants produce ROS and RNS as a defense response when they perceive an infectious stimulus from a pathogen [8,9]. The generated ROS and RNS function as signaling molecules that contribute to the activation of plant immunity [8,10]. The functional ROS produced by plant cells include superoxide anion (O 2 − ), hydroxyl radical (OH), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ), among others. O 2 − and H 2 O 2 have been of particular interest in studies of the mechanisms of plant disease defense [11]. Also, RNS such as NO are produced during plant immune responses [12,13]. Plant hormones such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are generally known to play important roles in the regulation of plant immunity [14], and many studies have reported that ROS and RNS signals are linked to the activities of these plant hormone signals [15]. Some reports indicate that exogenous application of RNS enhances plant disease resistance. Treatment with NO-releasing compounds appears to suppress tobacco mosaic virus infection in tobacco and rice black-streaked dwarf virus infection in rice through salicylic acid-mediated resistance [16,17]. Furthermore, exposure of Arabidopsis to NO 2 gas enhances basal resistance to Botrytis cinerea and Pseudomonas syringae by activating SA and JA signaling [18]. Exposure to high concentrations of NO 2 gas, but not NO gas, induces cell death in A. thaliana [19]. When used to treat plants, NO 2 gas is known to convert to nitrate (NO 3 − ) and nitrite (NO 2 − ). Thus, NO 3 − , NO, and H 2 O 2 might act in a coordinated manner to regulate NO 2 -induced cell death [19]. Other reports show that NO and NO 2 induce protein S-nitrosylation and tyrosine nitration, thereby regulating protein functions such as plant immunity and cell death [9,20,21].
Dinitrogen pentoxide is an active nitrogen species whose physiological effects are poorly understood due to difficulties with its synthesis and storage. We have developed a device that uses plasma technology to generate an active species gas containing a high concentration of N 2 O 5 [6]. The gas generated using the plasma technology, which we call "N 2 O 5 gas" in this study, contains a certain amount of O 3 and NO 2 in addition to its high concentration of N 2 O 5 due to the nature of the matter. However, because the N 2 O 5 gas we generate using this system has unique properties that cannot be created using conventional technology at present, it is meaningful to gain insight into its physiological effects. Although N 2 O 5 readily dissolves in water and results in nitric acid (HNO 3 ) via reactive intermediates [22], the effects on plants of such N 2 O 5 -induced chemical reactions are unknown. Moreover, there is currently no knowledge of plant responses after exposure to N 2 O 5 gas. In the present study, we exposed Arabidopsis to plasma-generated N 2 O 5 gas and analyzed its effects on disease resistance and post-exposure changes in gene expression after the exposure in order to elucidate the effects of N 2 O 5 gas on plant immune responses.

Dinitrogen pentoxide gas generation from air
Dinitrogen pentoxide gas, which is highly reactive, not storable under ambient conditions, and not typically available in the gas market, was prepared by an atmospheric pressure plasma device, developed recently in our previous work as shown in S1A Fig [6]. This device, which has electric controls designed for plant exposure experiments, can produce a continuous supply of N 2 O 5 gas from only compressed air via a chemical reaction chain involving N 2 O 5 , NO 2 , and O 3 . Importantly, to prevent contamination by any chemicals, no chemical compounds were used for the generation N 2 O 5 gas by the given plasma device. Furthermore, exactly the same conditions without reactive species were created by turning off the generation of air plasmas by the plasma device. Due to the unavoidable generation and decomposition reaction chains involving N 2 O 5 under physiological conditions, N 2 O 5 selectivity is limited up to 10 at a density of approximately 240 ppm, which allows simultaneously high density and high selectivity at room temperature. The minor gas components of O 3 , NO 2 , N 2 O, and HNO 3 that we measured that were unavoidably present in the N 2 O 5 gas are summarized in S1C Fig [6]. This N 2 O 5 gas mixture synthesized from air is henceforth described in this paper simply as N 2 O 5 gas.

Plant cultivation and N 2 O 5 gas exposure of plants
Wild-type plants of Arabidopsis thaliana ecotype Columbia (Col-0) and the mutants coi1-1, ein2-1, and npr1-1 in the same background were sown on soilless mix (Metro-Mix 350, San Gro, Canada) and grown for 2 weeks. Each seedling was then transferred to a new pot for further cultivation for 3 weeks in a growth chamber under short day conditions (light 10 h/dark 14 h) at 23˚C. Homozygous coi1-1 plants were screened using a dCAPs marker before transplanting [23]. The N 2 O 5 gas generated by the transportable plasma device was used for exposing plants to N 2 O 5 gas [6]. The plants were incubated for 30 min under a clear cover prior to exposure to N 2 O 5 gas to ensure uniform humidity conditions. Dry air containing N 2 O 5 at a density of approximately 240 ppm was emitted at 2 L/min from the outlet of a polytetrafluoroethylene (PTFE) tube with a 4-mm inner diameter. Each A. thaliana pot was placed 5 cm downstream from the N 2 O 5 gas outlet tube for 20 s once per day with an outer plastic shroud to prevent room air flow disturbances from causing unexpected processes as shown in S1B Fig.

Botrytis cinerea inoculation
Botrytis cinerea isolated from Brassica species (MAFF 237695) was provided from NARO Gen-eBank [24]. Botrytis Cinerea was cultured on potato dextrose agar (Difco, Detroit, MI) medium at 23˚C for 3 days under dark conditions, and then plates were transferred to incubate under continuous black light (FL10BLB; Toshiba Corp., Tokyo, Japan) for 3 days to induce conidia formation. Conidia were suspended in potato dextrose broth (Difco) using a paint brush and then filtered through four layers of gauze. Conidial suspensions were centrifuged at 400 × g for 5 min and the supernatant was removed. The collected conidia were resuspended in 1/8 diluted potato dextrose broth to a concentration of 2 × 10 5 conidia/mL. Five-week-old plants were exposed to N 2 O 5 gas once per day for 3 days, and the plants were inoculated with B. cinerea at 24 h after the final N 2 O 5 gas exposure. A separate set of control plants were sprayed with 200 μM methyl jasmonate (MeJA) and incubated for one day under a clear cover for comparison with the effect of the N 2 O 5 gas. The conidial suspension (5 μL) was spotted onto a fully expanded leaf and incubated for 2 days while maintaining high humidity. The area of each lesion (mm 2 ) was measured to evaluate disease severity. Trypan blue staining was performed to detect dead cells, as previously described [25]. Statistical analyses were performed using Student's t-test or the Tukey-Kramer test, depending on the number of experimental groups. Each experiment was performed at least twice and similar results were obtained each time.

Inoculation with Pseudomonas syringae pv. tomato DC3000
Five-week-old plants were exposed to N 2 O 5 gas once per day for 3 days, and then Pseudomonas syringae pv. tomato DC3000 (Pst) was inoculated at 24 h after the final gas exposure. King's B liquid medium supplemented with 50 μg/mL of rifampicin was used for Pst culture at 25˚C for 1 day and the bacterial concentration was adjusted with 10 mM MgCl 2 solution to an OD 600 value of 0.002. The bacterial suspension was infiltrated into the intercellular spaces of three fully expanded leaves per plant using a syringe. Mock treatment was performed by infiltration with 10 mM MgCl 2 in the absence of bacteria. The inoculated leaves were sampled and ground using a pestle in a tenfold volume of sterile water at 2 days after inoculation. Successive dilutions of the ground leaf tissue were spread onto King's B solid medium supplemented with rifampicin (50 μg/mL), incubated at 25˚C for 2 days, and the number of Pst colonies formed was counted. In addition, bacterial biomass was assessed by calculating the ratio of bacterial DNA to plant DNA using qPCR. Inoculated leaves were collected at 0, 1, 2 and 3 days after infection and total DNA was extracted using an ISOPLANT II kit (Nippon Gene Co., Tokyo, Japan) according to the manufacturer's protocols. To quantify plant DNA and Pst DNA, RHIP1 and OprF sequences [26], respectively, were amplified by qPCR using TB Green 1 Premix Ex Taq™ II (Takara Bio Inc., Shiga, Japan) on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) (S1 Table). Student's t-test was performed for statistical analysis and no significant differences were found. Each experiment was performed three times with similar results.

Inoculation with cucumber mosaic virus
Cucumber mosaic virus strain yellow (CMV(Y)) was inoculated and propagated on Nicotiana benthamiana plants and purified as described [27]. Five-week-old Arabidopsis plants were exposed to N 2 O 5 gas once per day for 3 days, and were inoculated with CMV(Y) at 24 h after the final N 2 O 5 gas exposure. Three leaves of the Arabidopsis plants were inoculated with CMV (Y) as described [28]. Briefly, mechanical inoculation of the virus was carried out onto leaves sprinkled with carborundum by rubbing the surface lightly with a cotton swab soaked in virus solution. Air exposure was used as control and was also subjected to CMV(Y) inoculation or mock treatment (water). An enzyme-linked immunosorbent assay (ELISA) was performed as described previously to quantify CMV(Y) multiplication [29]. A rabbit antibody against the CMV(Y) coat protein (CP) and alkaline phosphatase-conjugated anti-rabbit IgG (Fc) (Promega, Madison, WI) were used as the primary and secondary antibodies, respectively. The compound p-nitrophenyl phosphate (1 mg/mL) in AP9.5 buffer (10 mM Tris-HCl [pH 9.5], 100 mM NaCl, 5 mM MgCl 2 ) was used as the substrate for alkaline phosphatase. The absorbance of the resulting phenolate solution was measured at 405 nm. The amount of CP in 0.025 mg total protein was calculated as average absorbance ± standard deviation. Student's t-test was performed for statistical analysis. Each experiment was performed at least twice with similar results.

Transcriptome analysis by RNA-seq
Five-week-old plants were exposed to N 2 O 5 gas for 20 s once per day for 3 days, and fully expanded leaves were sampled at 24 h after the final exposure to N 2 O 5 gas (hereafter, N 2 O 5gas-exposed). Plants exposed to air served as the control experiment (hereafter, Air-control). Total RNA was extracted from leaves using the TRIzol method [30]. Macrogen Japan on the Illumina platform was used for RNA-seq analysis in order to obtain 151-bp paired-end sequences. Fifty-one M reads were obtained for the Air-control, and 46 M reads for the N 2 O 5gas-exposed sample were obtained. Differentially expressed gene analysis was performed between the Air-control and N 2 O 5 -gas-exposed samples. Fold changes (fc) in transcript abundances were calculated using exactTest in the edgeR package [31] for each sequence pair comparison. Significant results are indicated with |fc|�2 at an exactTest raw p-value <0.05. Functional category enrichment was defined by implementing the Gene Ontology (GO) tool online (http://geneontology.org/). The data for RNA-Seq have been deposited in the DDBJ Sequence Read Archive (DRA) (https://www.ddbj.nig.ac.jp/dra/index-e.html) and are accessible through DRR Run accession numbers: DRR345814 and DRR345815.

Gene expression analysis by qRT-PCR
Total RNA was extracted from individual seedlings using the TRIzol method [30]. Reverse transcription and subsequent PCR were performed using PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara Bio Inc.). The relative abundances of mRNA transcripts for each gene of interest were determined by qRT-PCR using TB Green 1 Premix Ex Taq™ II (Takara Bio Inc.) and a 7300 Real-Time PCR system (Applied Biosystems). Transcript abundance was calculated and represented as fold difference relative to the abundance of ACTIN2 transcripts. The average and standard deviation of values of three independent seedlings were then calculated. Student's t-test, Dunnett's test, or Tukey-Kramer test were performed for statistical analysis depending on the number and character of experimental groups. Each experiment was performed at least twice with similar results. Primers used in this study are listed in S1 Table.

Statistical analysis
All data were subjected to analysis of variance and various post-hoc tests using R version 3.6.3 (R Foundation for Statistical Computing, Vienna, Austria).

Determination of N 2 O 5 gas exposure conditions for Arabidopsis thaliana
To determine the N 2 O 5 gas exposure conditions that would not be harmful for plant growth, A. thaliana plants were exposed to N 2 O 5 gas by placing them 5 cm downstream from the gas outlet tube for 0 s, 10 s, 20 s, 30 s, 40 s, 60 s, 2 min, or 5 min. Immediately after the N 2 O 5 gas exposure for 300 s, leaves turned brown and died by 24 h after exposure (Fig 1). Clear injury was observed with N 2 O 5 gas exposure of 40 s at 6 h post-exposure (Fig 1). However, no apparent change in leaves was observed with up to 30 s of N 2 O 5 gas exposure even at 4 days after exposure (Fig 1). We have also confirmed that N 2 O 5 gas exposure for 20 s repeated once per day for 3 days did not cause apparent injury to plants. Therefore, exposure to N 2 O 5 gas for 20 s was chosen as the experimental treatment for the following analyses.
Effects of the N 2 O 5 gas exposure on disease resistance Air-exposed and N 2 O 5 -gas-exposed plants were inoculated with B. cinerea. The lesions were smaller on the N 2 O 5 -gas-exposed plants than on the control plants at 2 days post-inoculation Red arrowheads indicate a leaf that turned brown immediately after the N 2 O 5 gas exposure. Yellow arrowheads indicated the location of leaves that were damaged by the N 2 O 5 gas. Scale bar is 2 cm. (Fig 2). Trypan blue staining also confirmed that the area of dead tissue caused by B. cinerea infection was smaller in the N 2 O 5 -gas-exposed plants than in the control plants (Fig 2A). Measurements of lesion diameter confirmed that N 2 O 5 -gas-exposed plants reduced lesion size to approximately 53% of the control. Furthermore, the size of lesions on the N 2 O 5 -gas-exposed plants was similar to that on the MeJA-treated plants (Fig 2A). Therefore, N 2 O 5 gas exposure appears to enhance the B. cinerea resistance of A. thaliana to the same extent as does MeJA.
Next, separate sets of N 2 O 5 -gas-exposed and air-exposed plants were inoculated with Pst. Chlorosis was clearly observed on the inoculated leaves of both N 2 O 5 -gas-exposed and airexposed plants at 3 days post-inoculation (Fig 3A). Analysis of bacterial growth by culture methods showed no effect of the N 2 O 5 gas exposure after 2 days of inoculation (Fig 3B). Furthermore, the results of qPCR analysis of bacterial biomass over time showed no effect of exposure to N 2 O 5 gas on bacterial growth (Fig 3C). It was observed that the growth of Pst was intense up to 2 days post-inoculation and reached saturation by 3 days post-inoculation in both treatments. These results indicate that Pst resistance is not enhanced by the N 2 O 5 gas exposure.
Analysis of CMV resistance in the N 2 O 5 -gas-exposed plants was carried out. Two days after CMV inoculation, virus propagation in inoculated leaves was quantified by ELISA using anti-CMV CP antibody. The accumulation of CMV CP was significantly reduced in the N 2 O 5 -gasexposed plants compared with air-exposed plants (Fig 4), which suggests that N 2 O 5 gas exposure enhances CMV resistance.

Changes in gene expression after exposure to N 2 O 5 gas
To evaluate changes in global gene expression caused by exposure to N 2 O 5 gas, RNA-Seq analysis was performed using N 2 O 5 -gas-exposed and air-exposed leaf samples at 24 h following the third exposure to N 2 O 5 gas. The transcripts of 828 genes had increased in abundance by more than twofold after exposure to N 2 O 5 gas (S2 Table), and the transcript abundances of 871 genes had decreased by less than half (S3 Table). The expression of several selected genes was checked by qRT-PCR and similar results were obtained (S2 Fig). Gene ontology term analysis of the genes exhibiting increased transcript abundance suggested that JA-and ET-dependent signaling pathways and disease resistance including systemic acquired resistance are activated by exposure to N 2 O 5 gas (S3A Fig). Although GO term analysis showed that responses to SA and abscisic acid were suppressed by exposure to N 2 O 5 gas (S3B Fig), the expression of PR1, a marker gene for responses involving SA, was induced (S2 Table).
Among the genes exhibiting increased transcript abundance, we chose to further analyze changes in expression over time after N 2 O 5 gas exposure of the defense-related genes PAD3 and PDF1.2 and transcription factors (TFs) WRKY26 and ORA59, which may be involved in JA and ET responses. Arabidopsis plants were exposed to air (control) or N 2 O 5 gas once for 20 s, and shoots of each individual plant were collected as independent samples for qRT-PCR at 1, 3, 6, 12, 24, and 48 h after exposure. Expression of PAD3 and WRKY26 transcripts was transiently induced within 3 h after N 2 O 5 gas exposure (Fig 5). Protein encoded by PAD3 gene is a cytochrome P450 involved in the biosynthesis of the antimicrobial compound camalexin from tryptophan (S6A Fig). Similarly, expression of transcripts of CYP71A12, CYP71A13, PEN2, and NIT2, which are involved in tryptophan metabolism (S6A Fig), was also induced (Fig 5  and S4 Fig), suggesting that tryptophan metabolism, including synthesis of camalexin and indole-glucosinolate derivatives, is enhanced by exposure of plants N 2 O 5 gas. It has also been reported that the functions of WRKY26 are redundant with those of the homologous WRKY25 and WRKY33 [32]. We also confirmed that the gene expression of WRKY25 and WRKY33 showed similar changes in gene expression as WRKY26 after N 2 O 5 gas exposure (Fig 5). These TFs might coordinate regulation of the response to treatment of plants with N 2 O 5 gas.  Meanwhile, ORA59 showed biphasic induction of expression at around 3 h and 24 h after N 2 O 5 gas exposure (Fig 5). The expression of PDF1.2, which encodes an antimicrobial peptide, gradually increased from 3 h after N 2 O 5 gas exposure and remained at a high level until at least 48 h later (Fig 5). In contrast, the expression of VSP2, a gene specifically inducible by JA, was slightly responsive to N 2 O 5 gas exposure with a significant but low level of induction at 12 h after N 2 O 5 gas exposure (S4 Fig). In addition, although the expression of PR1 was induced at 12 to 24 h after N 2 O 5 gas exposure, it decreased to its basal level after 48 h (S4 Fig).

Responses to N 2 O 5 gas in Arabidopsis mutants deficient in phytohormone signaling pathways
To determine whether the observed responses to N 2 O 5 gas exposure are mediated by the JA and ET signaling pathways, we exposed mutant plants for each of these phytohormone Scale bar indicates 1 cm. (B) Inoculated leaves were collected at 2 days after infection and the bacterial titer (cfu/mg fresh weight of leaf material) was determine by the colony counting method. The graph shows means (± standard deviation) of the results from three independent plants. No significant differences between the treatments were detected (Student's t-test, P < 0.05). (C) Plant biomass and bacterial biomass were calculated by qPCR and the ratio of bacterial DNA per plant DNA was shown. There were no significant differences between treatments at each time point (Student's t-test, n = 3, P < 0.05). https://doi.org/10.1371/journal.pone.0269863.g003

Fig 4. Induction of cucumber mosaic virus resistance in
Arabidopsis plants by N 2 O 5 gas exposure. Arabidopsis plants exposed to N 2 O 5 gas were inoculated with CMV(Y). Two days after inoculation, the inoculated leaves were harvested and subjected to enzyme-linked immunosorbent assay using an antibody against the CMV CP. Asterisks denote significant differences (Student's t-test, n = 6, P < 0.05).
https://doi.org/10.1371/journal.pone.0269863.g004 signaling pathways to N 2 O 5 gas and analyzed their responses. Arabidopsis plants were exposed to air (control) or N 2 O 5 gas for 20 s, and shoots of each individual plant were harvested at 2 and 24 h later for qRT-PCR The induction of WRKY33, WRKY26, PEN2, and PAD3 expression was reduced in coi1-1, a JA signaling mutant at 2 h after N 2 O 5 gas exposure, whereas expression of these genes was similar to the wild type in ein2-1, an ET signaling mutant (Fig 6  and S5 Fig). The transcript abundance of ORA59 was relatively lower in plants carrying either mutation especially ein2-1, at 2 h after N 2 O 5 gas exposure. The transcript abundance of ORA59 was significantly lower only in coi1-1 at 24 h after exposure to N 2 O 5 gas. The contribution of ET and JA to the regulation of ORA59 expression seems to differ between 2 h and 24 h after N 2 O 5 gas exposure (Fig 6). Induction of the expression of PDF1.2 was greatly attenuated in both the coi1-1 and ein2-1 mutants, especially at 24 h after N 2 O 5 gas exposure (Fig 6). These results indicated that both JA and ET signaling have important roles in the activation of gene expression by N 2 O 5 gas.
In addition, we analyzed the induction of disease resistance by exposure to N 2 O 5 gas in coi1-1 and ein2-1 mutants. The SA signaling mutant npr1-1 was also used in these experiments. Wild-type, coi1-1, ein2-1, and npr1-1 plants were exposed to N 2 O 5 gas under the same conditions as in Fig 2. The sizes of lesions caused by B. cinerea infection were reduced with exposure to N 2 O 5 gas as compared with the Air-control in ein2-1 and npr1-1 but were not significantly different in coi1-1 as shown in Fig 7A. These results indicated that JA signaling has a major role in the enhancement of B. cinerea resistance by N 2 O 5 gas. In contrast, the induction of CMV resistance by exposure to N 2 O 5 gas was compromised in ein2-1 and npr1-1, but was maintained in coi1-1 (Fig 7B). In particular, the effect of the N 2 O 5 gas tended to be weak in ein2-1, suggesting that ET may be a major factor in CMV resistance induced by N 2 O 5 gas.

Discussion
In this study, we showed that exposure of Arabidopsis thaliana to N 2 O 5 gas produced from airderived plasma can activate plant immunity mainly through JA and ET signaling. Specifically, we showed that exposure of plants to N 2 O 5 gas enhanced B. cinerea and CMV resistance but not Pst resistance. However, it is important to note that although N 2 O 5 gas is composed mainly of highly concentrated N 2 O 5 , it also contains a certain amount of the other active species such as O 3 and NO 2 (S1C Fig). Methods for exogenously exposing plants to ROS or RNS in a gaseous state for disease control have been reported using O 3 and NO 2 [18,33]. Increased production of SA, ET, and JA has been reported upon O 3 exposure in A. thaliana [34][35][36]. Ethylene is thought to be involved in O 3 sensitivity because O 3 -induced damage is reduced in ethylenedeficient mutants. However, ET production is suppressed and damage is reduced in MeJAtreated plants after O 3 exposure, suggesting that JA acts antagonistically with ET [34]. With regard to SA, the accumulation of SA after O 3 exposure was high in the ET-overproducing mutant eto1, whereas ET production was low in the SA-deficient mutant, suggesting that ET and SA work cooperatively after O 3 exposure [35]. Ozone exposure also effectively inhibits the propagation of several plant viruses such as tobacco mosaic virus and soybean mosaic virus [37,38]. In the present study, the N 2 O 5 gas exposure shown to enhance CMV resistance was dependent mainly on ET signaling pathways according to our analysis using phytohormone signaling mutants (Fig 7B). Ethylene signaling is also partially involved in the CMV resistance  . The mRNA transcript abundances of genes related to plant disease defense, including  WRKY25, WRKY26, WRKY33, PEN2, CYP71A13, PAD3, ORA59, and PDF1.2, were analyzed using qRT-PCR. Asterisks denote significant differences to 0 h samples (without N 2 O 5 gas exposure; Dunnett's test, n = 3, P < 0.05). ACT2, ACTIN2.

PLOS ONE
conferred by RCY1, a disease-resistance protein in A. thaliana [39]. The CMV resistance induced by N 2 O 5 gas and that conferred by RCY1 might employ a common ET-mediated mechanism. Meanwhile, although activation of SA responses was not detected during the GO term analysis of our RNA-Seq results, transient activation of PR1 expression was confirmed (S3B and S4 Figs and S2 Table). These results imply that SA signaling may also be partially activated. The fact that there was no significant enhancement of CMV resistance by N 2 O 5 gas exposure in the npr1-1 mutant supports this possibility (Fig 7B). Because a cooperative function of ET and SA has also been reported for O 3 [35], it is possible that O 3 contained in the N 2 O 5 gas partially contributes to the CMV resistance induced by the N 2 O 5 gas. Otherwise, N 2 O 5 has a strong oxidative effect much like that of O 3 , so an unknown mechanism might be operating due to an oxidative effect of N 2 O 5 . Further analysis is needed to understand the mechanistic details of the enhancement of CMV resistance by N 2 O 5 gas.
Botrytis cinerea resistance is regulated by a complex network of SA, ET, and JA signals [40]. However, JA and ET play major roles in resistance to necrotrophic pathogens including B. cinerea [41]. An increase in SA content has been observed immediately after exposure in the case of exposure to NO 2 gas [18]. Interestingly, gene expression related to the synthesis and metabolism of JA is also activated after exposure to NO 2 gas. Also, a reduction in the amount of active JA and accumulation of its metabolites is observed [18]. Exposure to NO 2 gas enhances B. cinerea resistance, but this effect is impaired in both SA-deficient NahG plants and JA biosynthetic mutants, suggesting that NO 2 -induced resistance enhancement involves not only SA accumulation but also activation of JA metabolism [18]. In contrast, our results suggest that N 2 O 5 gas enhances resistance to B. cinerea mainly by activating JA signaling (Figs 2 and 7A). Gene expression analysis suggests that JA and ET signals are activated in a coordinated manner by exposure to N 2 O 5 gas (S3A Fig). Analysis using phytohormone signaling mutants also supports that JA and ET signals are activated by exposure to N 2 O 5 gas (Fig 6). However, GO term analysis of our RNA-Seq results suggests that SA signaling was either not activated or was somewhat suppressed by the exposure of plants to N  In general, SA plays a central role in resistance to Pst, while JA and ET act antagonistically [42,43]. However, JA is reported to play a cooperative role with SA in the induction of Pst resistance by oligogalacturonides and chitosan oligosaccharides [44,45]. In the present study, we did not observe any enhancement of Pst resistance by the N 2 O 5 gas (Fig 3), which might have been because SA signaling was not predominantly activated compared with JA (S3 Fig). Because NO 2 -gas exposure enhances Pst resistance via SA signaling [18], the differential effects of N 2 O 5 gas and NO 2 gas on disease resistance can be confirmed again.
A key transcription factor, WRKY33, controls the expression of genes regulating resistance to B. cinerea via ET/JA signaling and camalexin biosynthesis such as CYP71A12, CYP71A13, and PAD3 [46,47]. The transient increase in the abundance of WRKY33 transcripts by exposure N 2 O 5 gas suggests activation of WRKY33-mediated B. cinerea resistance (Fig 5). Because the N 2 O 5 gas induced the expression of CYP71A12, CYP71A13, PAD3, and PEN2 (Fig 5), it is likely that secondary metabolites derived from tryptophan such as camalexin, indole-glucosinolate derivatives, and indole-carboxylic acid (S6A Fig) are involved in the N 2 O 5 -gas-induced B. cinerea resistance. Furthermore, WRKY25 and WRKY26, which are functionally redundant with WRKY33 [32], are thought to regulate these metabolic systems in a coordinated manner (Fig 5). However, it has been reported that the induction of B. cinerea resistance by NO 2 is PAD3-dependent but not accompanied by an increase in camalexin content [18]. Further analysis is needed to determine which metabolites, including camalexin, contribute to the enhancement of B. cinerea resistance by N 2 O 5 gas. Meanwhile, although exposure to N 2 O 5 gas strongly induced the expression of ORA59 and PDF1.2 (Fig 5), the induction of VSP2 was relatively weak (S4 Fig). The expression of ORA59, which encodes a TF, is regulated by both ET and JA and controls the expression of PDF1.2, which is involved in disease resistance (S6B Fig). However, VSP2 is thought to function in wounding response and insect resistance under the control of the TF MYC2 in a JA-specific manner (S6B Fig) [14,48]. It is possible that ET signaling becomes more dominant in the relationship between ORA59 and MYC2 during exposure to N 2 O 5 gas.
Interestingly, the response of WRKY33 transcript to exposure to N 2 O 5 gas is highly similar to the responses to treatments with damage-associated molecular patterns (DAMPs) such as HMGB3 and Pep1 [49]. In plants and animals, DAMPs released from cells due to injury induce immune responses [50,51]. Therefore, exposure to N 2 O 5 gas causes slight cellular damage (Fig 1), which might result in the release of DAMPs into the apoplast of plant tissues. Proteinaceous DAMPs such as HMGB1 and HSPs are known to play an important role in the inflammatory response in animal cells [52]. Pattern recognition receptors (PRR, e.g., Toll-like receptor 4) recognize DAMPs released from damaged cells, and thereby transmit the damage stimulus to surrounding cells [52]. Furthermore, post-translational modifications of DAMPs are known to alter their functions [53]. A recent report indicates that proteinaceous DAMPs, in which tyrosine residues have been modified by nitration, activate the PRR more strongly than do unmodified DAMPs in HeLa cells [54]. Dinitrogen pentoxide is a powerful oxidizing and nitrating agent, and is an important agent widely used in the nitration and S-nitrosylation of organic compounds, as for the production of nitrotyrosine when tyrosine is treated with the N 2 O 5 gas [6]. Therefore, exposure to the N 2 O 5 gas might contribute not only to the release of DAMPs, but also to the modification of DAMPs by nitration or S-nitrosylation to influence their function in plant immunity. The involvement of DAMPs in the enhancement of disease resistance by N 2 O 5 gas will be elucidated in future analyses.
In conclusion, we have shown that N 2 O 5 gas has potential for development as a new technology for plant disease control. N 2 O 5 is converted to nitric acid by reacting with water, and it can be used by plants as a source of nitrogen. Therefore, treatment with N 2 O 5 gas would be almost free from risks of environmental pollution. In addition because the amount of electricity required for production of N 2 O 5 gas is relatively low [6], control of plant diseases using N 2 O 5 gas could contribute as a low-cost and environmentally friendly technology to the establishment of a sustainable agricultural system. Furthermore, the device used in this study can selectively supply O 3 or NO/NO 2 by mode switching [6]. Since the present study was performed under laboratory conditions using A. thaliana, validation under field conditions using crops is a subject for future work. However, this type of approach might be useful for efficiently controlling plant diseases by exposing crop species to the appropriate active gas composition for the type of disease. Recently, Kumar and co-workers reported that glycine betaine and Arbuscular mycorrhizal fungi treatment reduces chromium toxicity via reduction of oxidative stress [55][56][57]. Combining such treatments with N 2 O 5 gas exposure may allow for the development of more effective and harmless disease control methods. To confirm the results of RNA-Seq, five genes with elevated transcript expression after exposure to N 2 O 5 gas were chosen for further analysis of their relative transcript abundances by qRT-PCR. Arabidopsis plants were exposed to air (control) or N 2 O 5 gas for 20 s once a day for 3 days. Shoots of each individual plant were collected as independent samples at 24 h after the third exposure. Total RNA was extracted and subjected to analysis of the relative mRNA transcript abundances of defense-related genes, including PDF1.2, PDF1.4, ORA59, WRKY26, and PR4. Data were normalized to ACTIN2 mRNA transcript abundance. Asterisks denote significant differences relative to the air control (Student's t-test, n = 3, P < 0.05). ACT2, ACTIN2. The fold change (N 2 O 5 /Air) calculated from qRT-PCR was compared to the fold change obtained from RNA-seq. (PDF)  Table. Genes whose transcript abundance increased more than twofold after exposure to N 2 O 5 gas compared to air control in RNA-seq analysis. (XLSX) S3 Table. Genes whose transcript abundance decreased less than half after exposure to N 2 O 5 gas compared to air control in RNA-seq analysis.